At least half of the world population shows evidence for past or present infection by hepatitis B virus (HBV), and the approximately 200 million carriers in the world today are at serious risk to chronic liver disease and, possibly, primary liver cancer. The classic marker for chronic infection by this virus is the surface antigen, HBsAg, which circulates in the serum of HBV carriers in three morphological forms: 22 nm spherical particles, 22 nm filaments of various lengths and the 42 nm spherical form known as the Dane particle. The 22 nm particles and filaments are subviral forms containing two predominant polypeptides, with apparent molecular weights of about 23,000 and 29,000, together with several minor polypeptides of larger size. The two predominant species, which are probably identical except that the larger is glycosylated, carry both the group (a) and the subtype (d/y) antigenic determinants of HBsAg. The Dane particle, which represents the infectious virion, consists of a lipoprotein coat (HBsAg) surrounding an internal core particle which contains a DNA polymerase and the 3200 base pair (bp) circular DNA genome. The 22 nm particle is the predominant form in the sera of chronic carriers and circulates at concentrations as high as 100-200 .mu.g/ml.
Characterization of the life cycle and biology of HBV has been hampered by its narrow host range, which is restricted to humans and a few other primates, and by its inability to grow in cultured cells. Problems associated with conventional purification and isolation from human serum are discussed, for example, in U.S. Pat. Nos. 4,113,712; 4,138,287 and 4,186,193.
Recently, however, several groups have succeeded in cloning the viral genome in Escherichia coli phage lambda (Charnay et al, Proc. Natl. Acad. Sci. USA 76, 2222-2226, 1979) and plasmid vector (Burrell et al, Nature 279, 43-47, 1979, and Sninsky et al, Nature 279, 346-348, 1979) and in determining its primary structure (Valenzuela et al, Nature 280, 815-819, 1979; Pasek et al, Nature 282, 575-579, 1979; and Galibert et al, Nature 281, 646-650, 1979). This has allowed the identification of a continuous 892 bp sequence that could encode surface antigen, a 549 bp sequence that may specify the core antigen and several additional open sequences of unknown function.
Although the DNA sequence provides crucial structural information, it clearly is not sufficient to establish all of the HBV gene products nor to understand how these products interact during infection of the target cell. For this purpose it would be useful to develop a system for introducing defined portions of the viral genome into cultured cells. Simian virus 40 (SV40), a small DNA tumor virus that can lytically infect cultured monkey cells, provides a useful vector for this purpose. The present invention provides for the construction and propagation of an SV40 recombinant carrying a 1350 bp fragment of HBV DNA that includes the structural sequences for surface antigen. Monkey kidney cells infected with this recombinant synthesize surface antigen that is excreted into the culture media as 22 nm particles. These results set an upper limit on the amount of HBV genetic information required for 22 nm particle formation and demonstrate the feasibility of using SV40 recombinants to study HBV gene expression in cultured primate cells. Ultimately, the antigen is useful in the preparation of vaccines, as a standard antigen reagent for testing hepatitis B surface antigen and antibody and as an antigen for immunizing animals to obtain a highly specific and strong antibody. Because the antigen is derived from a single cloned gene, it is homogeneous. Moreover, this unique process is free of contaminating serum components that conventional human serum processing cannot avoid.